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Three Gram-negative, non-pigmented, rod-shaped, facultatively aerobic bacterial strains, designated d8-1T, d8-2 and IMCC1716, were isolated from a freshwater spring sample and a eutrophic freshwater pond. Based on characterization using a polyphasic approach, the three strains showed highly similar phenotypic, physiological and genetic characteristics. All of the strains harboured the nitrogenase gene nifH, but nitrogen-fixing activities could not be detected in nitrogen-free culture media. The three strains shared 99.6–99.7 % 16S rRNA gene sequence similarity and showed 89–100 % DNA–DNA relatedness, suggesting that they represent a single genomic species. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains d8-1T, d8-2 and IMCC1716 formed a monophyletic branch in the periphery of the evolutionary radiation occupied by the genus Azonexus. Their closest neighbours were Azonexus caeni Slu-05T (96.7–96.8 % similarity) and Azonexus fungiphilus BS5-8T (96.3–96.6 %). The DNA–DNA relatedness of the novel strains to these two species of the genus Azonexus was less than 70 %. The isolates could also be differentiated from recognized members of the genus Azonexus on the basis of phenotypic and biochemical characteristics. It is evident, therefore, that the three strains represent a novel species of the genus Azonexus, for which the name Azonexus hydrophilus sp. nov. is proposed. The type strain is d8-1T (=LMG 24005T=BCRC 17657T).
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International Journal of Systematic and Evolutionary Microbiology vol. 58 , part 4, pp. 946 - 951
Supplementary Fig. S1. nifH gene sequence-based phylogenetic tree generated by using the neighbour-joining method, showing the relationships between strains d8-1 T, d8-2, IMCC 1716 and other nitrogen fixing bacteria.
Supplementary Fig. S2. Electron micrograph of cells of Azonexus hydrophilus strain d8-1 T.
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